Sanglifehrins
Sanglifehrin A (SfA), 5 and its natural congeners belong to a class of mixed non-ribosomal peptide/polyketides, produced by Streptomyces sp. A92-308110 (also known as DSM 9954) (see WO 97/02285 and WO 98/07743), which were originally discovered on the basis of their high affinity to cyclophilin A (CypA). SfA is the most abundant component in fermentation broths and exhibits approximately 20-fold higher affinity for CyPA compared to Cyclosporine A (CsA), 1. This has led to the suggestion that sanglifehrins could be useful for the treatment of HCV (WO2006/138507). Sanglifehrins have also been shown to exhibit a novel mechanism of immunosuppressive activity as compared to CsA (Sanglier et al., 1999; Fehr et al., 1999). SfA binds with high affinity to the CsA binding site of CyPA (Kallen et al., 2005).
Biosynthesis of Sanglifehrins
Sanglifehrins are biosynthesised by a mixed polyketide synthase (PKS)/Non-ribosomal peptide synthetase (NRPS) (see WO2010/034243). The 22-membered macrolide backbone consists of a polyketide carbon chain and a tripeptide chain. The peptide chain consists of one natural amino acid, valine, and two non-natural amino acids: (S)-meta-tyrosine and (S)-piperazic acid, linked by an amide bond. Hydroxylation of phenylalanine (either in situ on the NRPS or prior to biosynthesis) to generate (S)-meta-tyrosine is thought to occur via the gene product of sfaA.
Semisynthetic Sanglifehrins
Examples of the generation of semisynthetic derivatives of natural sanglifehrins have been described in the literature. These include sangamides (Moss et al., 2011, WO2011/098809), ester macrocyclic analogues of sanglifehrin (WO2011/098805) and ketone macrocyclic analogues of sanglifehrin (WO2011/098808). One of the cited reasons for generation of analogues has been to improve oral bioavailability. Other analogues have also been described in the literature (e.g. Sedrani et al., 2003, WO 2006/138507, Gaither et al., 2010).
Uses of Sanglifehrins
Immunosuppressive Action of Sanglifehrins
The immunosuppressive mechanism of action of SfA is different to that of other known immunophilin-binding immunosuppressive drugs such as CsA, FK506 and rapamycin. SfA does not inhibit the phosphatase activity of calcineurin, the target of CsA (Zenke et al. 2001), instead its immunosuppressive activity has been attributed to the inhibition of interleukin-6 (Hartel et al., 2005), interleukin-12 (Steinschulte et al., 2003) and inhibition of interleukin-2-dependent T cell proliferation (Zhang & Liu, 2001). However, the molecular target and mechanism through which SfA exerts its immunosuppressive effect is hitherto unknown.
The molecular structure of SfA is complex and its interaction with CyPA is thought to be mediated largely by the macrocyclic portion of the molecule. In fact, a macrocyclic compound (hydroxymacrocycle, 6) derived from oxidative cleavage of SfA has shown strong affinity for CyPA (Sedrani et al., 2003). X-ray crystal structure data has shown that the hydroxymacrocycle binds to the same active site of CyPA as CsA. Analogues based on the macrocycle moiety of SfA have also previously been shown to be devoid of immunosuppressive properties (Sedrani et al., 2003), providing opportunity for design of non-immunosuppressive CyP inhibitors for potential use in HCV and HIV therapy.
Converse to this, there is also an opportunity to develop immunosuppressive agents with low toxicity for use in such areas as prophylaxis of transplant rejection, autoimmune, inflammatory and respiratory disorders, including, but not limited to, Crohn's disease, Behcet syndrome, uveitis, psoriasis, atopic dermatitis, rheumatoid arthritis, nephritic syndrome, aplastic anaemia, biliary cirrhosis, asthma, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD) and celiac disease. Sanglifehrins have been shown to have a novel mechanism of immunosuppressive activity (Zenke et al., 2001), potentially acting through dendritic cell chemokines (Immecke et al., 2011), and there is therefore an opportunity to develop agents with a mechanism of action different to current clinical agents, such as cyclosporine A, rapamycin and FK506.
Human Immunodeficiency Virus (HIV)
Cyclophilin inhibitors, such as CsA and DEBIO-025 have also shown potential utility in inhibition of HIV replication. The cyclophilin inhibitors are thought to interfere with function of CyPA during progression/completion of HIV reverse transcription (Ptak et al., 2008). However, when tested clinically, DEBIO-025 only reduced HIV-1 RNA levels ≧0.5 and >1 log 10 copies/mL in nine and two patients respectively, whilst 27 of the treated patients showed no reduction in HIV-1 RNA levels (Steyn et al., 2006). Following this, DEBIO-025 was trialled in HCV/HIV coinfected patients, and showed better efficacy against HCV, and the HIV clinical trials were discontinued (see Watashi et al., 2010).
Hepatitis B Virus
Hepatitis B is a DNA virus of the family hepadnaviridae, and is the causative agent of Hepatitis B. As opposed to the cases with HCV and HIV, there have been very few published accounts of activity of cyclophilin inhibitors against Hepatitis B virus. Ptak et al. 2008 have described weak activity of DEBIO-025 against HBV (IC50 of 4.1 μM), whilst Xie et al., 2007 described some activity of CsA against HBV (IC50>1.3 μg/mL). This is in contrast to HIV and HCV, where there are numerous reports of nanomolar antiviral activity of cyclophilin inhibitors.
Inhibition of the Mitochondrial Permeability Transition Pore (mPTP)
Opening of the high conductance permeability transition pores in mitochondria initiates onset of the mitochondrial permeability transition (MPT). This is a causative event, leading to necrosis and apoptosis in hepatocytes after oxidative stress, Ca2+ toxicity, and ischaemia/reperfusion. Inhibition of Cyclophilin D (also known as Cyclophilin F) by cyclophilin inhibitors has been shown to block opening of permeability transition pores and protects cell death after these stresses. Cyclophilin D inhibitors may therefore be useful in indications where the mPTP opening has been implicated, such as muscular dystrophy, in particular Ullrich congenital muscular dystrophy and Bethlem myopathy (Millay et al., 2008, WO2008/084368, Palma et al., 2009), multiple sclerosis (Forte et al., 2009), diabetes (Fujimoto et al., 2010), amyotrophic lateral sclerosis (Martin 2009), bipolar disorder (Kubota et al., 2010), Alzheimer's disease (Du and Yan, 2010), Huntington's disease (Perry et al., 2010), recovery after myocardial infarction (Gomez et al., 2007) and chronic alcohol consumption (King et al., 2010).
Further Therapeutic Uses
Cyclophilin inhibitors have potential activity against and therefore in the treatment of infections of other viruses, such as Varicella-zoster virus (Ptak et al., 2008), Influenza A virus (Liu et al., 2009), Severe acute respiratory syndrome coronavirus and other human and feline coronaviruses (Chen et al., 2005, Ptak et al., 2008), Dengue virus (Kaul et al., 2009), Yellow fever virus (Qing et al., 2009), West Nile virus (Qing et al., 2009), Western equine encephalitis virus (Qing et al., 2009), Cytomegalovirus (Kawasaki et al., 2007) and Vaccinia virus (Castro et al., 2003).
There are also reports of utility of cyclophilin inhibitors and cyclophilin inhibition in other therapeutic areas, such as in cancer (Han et al., 2009).
Oral Bioavailability of Sanglifehrins
One of the issues in drug development of natural and non-natural sanglifehrins is low oral bioavailability (e.g. see Gregory et al., 2011). This can lead to higher cost of goods, increased chance of food effect and higher interpatient variability. Whilst one route to improve this is to generate novel analogues, another route is to use formulations.
Therefore there remains a need to identify novel formulations for oral dosage of sanglifehrins, which can increase the oral bioavailability of this potentially important class of drug. Sanglifehrins may have utility in the treatment of HCV infection, but also in the treatment of other disease areas where inhibition of cyclophilins may be useful, such as HIV infection, muscular dystrophy or aiding recovery after myocardial infarction or where immunosuppression or anti-inflammatory effect is useful.